Human cells respond to external signals or stimuli in various ways through a signaling process. The signaling process usually includes multi-step biochemical reactions that are generally catalyzed by enzymes and messenger proteins for transferring the signal to the next step. This process often consists of activating a certain enzyme that acts on another enzyme to change the activity of the enzyme. This signaling process either results in a reaction that increases or reduces a certain metabolite or causes significant intracellular changes such as gene expression, cell division or cell death.
The most important reversible covalent modification in cell signaling is protein phosphorylation. A phosphate group (PO4) is reversibly incorporated into a protein by the action of kinase and phosphatase, and the enzymatic activity of the protein is changed due to the incorporated phosphate group. Phosphorylation on serine is the most common, followed by threonine and tyrosine residues. Phosphorylation of protein changes the structure of the protein, and as a result, influences the activity of the protein, the interaction with other proteins, the intracellular distribution of the protein, and the stability of the protein to regulate the function of the protein, thereby influencing cell signaling. Protein phosphorylation usually occurs such that about ⅓ of proteins that are expressed in about 25,000 human genes are phosphorylated. Thus, protein phosphorylation regulates all physiological activities, including cell signaling activity, in eukaryotic organisms including humans. Therefore, when abnormalities in protein phosphorylation are caused by mutations or the like, they cause various diseases such as cancer or neurological diseases. In order to understand cell signaling and elucidate the cause of various diseases attributable to abnormalities in signaling, technology of regulating protein phosphorylation and producing a large amount of a protein phosphorylated at a specific amino acid is essentially required. In addition, this method is necessary for elucidating the function of phosphorylated proteins and developing drugs that regulate the function of phosphorylated proteins related to diseases.
Protein phosphorylation that usually occurs in eukaryotic organisms is very rapid, reversible and multiple, and thus it is very difficult to produce a large amount of a protein, uniformly phosphorylated at a specific amino acid, using eukaryotic cells. In bacteria that are mainly used to produce large amounts of recombinant proteins, a protein phosphorylation mechanism does not exist, and thus it is impossible to produce a phosphorylated protein. Heretofore, various methods (WO 2012/048249, WO 2009/099073, WO 2006/107813, and JP 2008/061538) have been proposed to produce phosphorylated proteins, but among these methods, only the method disclosed in WO 2012/048249 can produce a serine-phosphorylated protein. However, this method has a shortcoming in that, because the efficiency of production of the phosphorylated protein is very low, the phosphorylated protein is produced only in an amount of ug per liter.
Accordingly, the present inventors have made extensive efforts to develop a method of producing a phosphorylated protein with significantly increased efficiency, and as a result, have found that, when a phosphorylated protein is produced using the SepRS and EF-Tu mutants selected by molecular evolution, it is produced in an amount of mg per liter, thereby completing the present invention. This method makes it possible to efficiently produce a protein with site-specific serine phosphorylation, which is one of the most abundant posttranslational modifications.